Isolation and characterization of β-transducin repeat-containing protein ligands screened using a high-throughput screening system

β-transducin repeat-containing protein (β-TrCP) is an F-box protein subunit of the E3 Skp1-Cullin-F box (SCF) type ubiquitin-ligase complex, and provides the substrate specificity for the ligase. To find potent ligands of β-TrCP useful for the proteolysis targeting chimera (PROTAC) system using β-TrCP in the future, we developed a high-throughput screening system for small molecule β-TrCP ligands. We screened the chemical library utilizing the system and obtained several hit compounds. The effects of the hit compounds on in vitro ubiquitination activity of SCFβ-TrCP1 and on downstream signaling pathways were examined. Hit compounds NPD5943, NPL62020-01, and NPL42040-01 inhibited the TNFα-induced degradation of IκBα and its phosphorylated form. Hence, they inhibited the activation of the transcription activity of NF-κB, indicating the effective inhibition of β-TrCP by the hit compounds in cells. Next, we performed an in silico analysis of the hit compounds to determine the important moieties of the hit compounds. Carboxyl groups of NPL62020-01 and NPL42040-01 and hydroxyl groups of NPD5943 created hydrogen bonds with β-TrCP similar to those created by intrinsic target phosphopeptides of β-TrCP. Our findings enhance our knowledge of useful small molecule ligands of β-TrCP and the importance of residues that can be ligands of β-TrCP.


Introduction
Ubiquitination of substrates occurs between ubiquitin and the target protein [1]. This type of modification affects many cellular processes, including growth factor-induced endocytosis, DNA repair, and chromatin modification/ transcriptional regulation [2]. E3 ligases are essential in the selection of substrates. Ubiquitin modification induces multiple types of cellular signaling; therefore, proper substrate selection ensures correct ubiquitin signaling [3]. The Skp1-Cullin-F box complex (SCF) is one of the classical types of E3 enzymes whose substrate specificity is determined by F-box proteins. As one of the F-box proteins, β-transducin repeat-containing protein (β-TrCP) is encoded by two different genes (FBXW1 and FBXW11), with indistinguishable biochemical identities in mammals [4,5]. β-TrCP, a substrate acceptor, along with CUL1, RING protein RBX1, and SKP1, forms the activated SCF β-TrCP E3 ligase, essential for regulating protein levels of many key targets [6]. It recognizes the consensus degradation motif DSGφXS disruption motif (φ and X represent hydrophobic and arbitrary amino acids, respectively). The binding of β-TrCP depends on the phosphorylation of the two serines of the disruption motif, which links phosphorylated signaling to ubiquitination and destruction of proteins [7].
Examining the IκBα ubiquitination using TR-FRET, GS143 was identified as an inhibitor of β-TrCP, which suppressed β-TrCP dependent IκBα ubiquitination. GS143 inhibited the TNF-α-induced IκBα degradation and the NF-κB downstream reactions, excluding p53 and β-catenin responses. It is a potent inhibitor of the NF-κB signaling pathway [8]. In this study, GS143 was used as a positive control.
In this study, we established a high-throughput screening system to screen β-TrCP ligands. Hit compounds were analyzed for their SCF β-TrCP inhibitory activities. The hit compounds may be used for PROTAC construction in the future.
In vitro ubiquitination assay Components of SCF complex (SKP1, Cul1, Rbx1, and hemagglutinin (HA)-tagged β-TrCP) were expressed in HEK293T cells. Anti-HA antibody was added into the SCF complexes expressed HEK 293T cell lysate and rotated at 4°C for 1 h. Aliquots of this mixture were added to protein G beads and rotated at 4°C for 30 min. After washing the beads in cell lysis buffer three times, the tube was centrifuged at 20,400 × g for 5 min and the supernatant was discarded. The protein on the beads was used as recombinant SCF β-TrCP complexes. Aliquots of SCF complexes bound on the beads were then added to ubiquitination reactions (10 mM Tris pH = 7.5, 5 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 20 mM creatine phosphate, 0.1 mg/mL creatine kinase, 5 mM NaF, 1 mM Na 3 VO 4 , 2 mg/mL ubiquitin, 0.5 μg E1 enzyme, 2 μg E2 enzyme) in a final volume of 30 µL. Compounds of 100 µM final concentration were added to reactions, and GST-Wee1KR was added as substrate 15 min later. The reaction mixtures were incubated at 37°C for 2 h and were further examined by immunoblotting.

Analysis of protein degradation
For examining the effect on degradation of IκBα, HeLa cells were cultured in the presence or absence of the compound for 30 min. TNF-α (Cat. No. TNA-H4211, Acro Biosystems, Newark, DE, USA) was added (40 ng/mL) and incubated for another 5 min. The cell extracts were analyzed using immunoblotting using antibodies against IκBα and phosphorylated IκBα (p-IκBα).
For examining the effect on degradation of β-catenin, HeLa cells were cultured in the presence or absence of the compound for 3 h. The cell extracts were examined by immunoblotting using antibodies against β-catenin and phosphorylated β-catenin (p-β-catenin).

Luciferase reporter assays
HeLa cells were seeded on 96-well plates. Subsequently, TCF and NF-κB transcription activity was examined by the luciferase assay system. T-cell Factor (TCF) transcription activity was assessed by TOP/FOP-Flash luciferase reporter assay, using a pRL-SV40 vector as an internal control. TOP FLASH contains three TCF transcription factors and a firefly luciferase reporter with a minimal promoter and an upstream of the luciferase reporter gene. FOP FLASH is similar to TOP reporter; however, it has three mutated TCF-binding sites and is used as a negative control. The average ratio of TOP to renilla luciferase divided by FOP was used as TCF activity. TOP flash (Sigma-Aldrich, Temecula, CA, USA) or FOP FLASH reporter plasmids (Sigma-Aldrich) and pRL-sv40 renilla luciferase reporter (Promega) were co-transfected into HeLa cells using Lipofectamine 3000 reagent kit (Thermo Fisher Scientific). Furthermore, 24 h after transfection, cells were cultured with the compounds of different dilutions for another 24 h. The cells were lysed, and the luminescence was measured using the Luciferase Assay System (Promega). A minimum of three samples were taken for each sample.
For NF-κB transcription activity, PGL-4.32 contains five copies of the NF-κB response element, which is responsible for the transcription of the luciferase reporter gene, with pRL-SV40 as the internal control. NF-κB transcription activity was determined by firefly luciferase expression and normalized by renilla luciferase level. pGL-4.32 reporter plasmids (Promega) and pRL-sv40 renilla luciferase reporter (Promega) were co-transfected into HeLa cells using Lipofectamine 3000 reagent kit. Subsequently, 24 h after transfection, cells were cultured with the compounds of different dilutions for another 24 h. Furthermore, TNF-α was added, and the plates were cultured for another 4 h. The cells were lysed, and the luminescence was measured using the Luciferase Assay System. A minimum of three samples were taken for each sample.

Molecular docking simulation
The 3D structures of the compounds were obtained from the NCBI PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The SDF file is transformed into PDB by PyMol software, and the Autodock tools-1.5.7 tool converts it into PDBQT format. The crystallographic data of β-TrCP1 (PDB code 1P22, PDB format) were obtained from Protein Data Bank and Chimera 1.16 software. The PDBQT files were created using the Autodock tool 1.5.7. Molecular docking simulation proceeded by executing the autodock vina algorithm. A total of 150 flexible docking runs, an energy range of 5, and 20 number modes were set, and the affinities (Kcal/mol) were calculated. Consequently, all the figures were made using PyMol software.

Statistical analysis
Luciferase reporter assay was examined in triplicate. The data were presented as the average ± S.D. GraphPad Prism 9.4.1 software (Macintosh Version by Software MacKiev 1994-2022 GraphPad Software, LLC) was used to evaluate the statistical differences among the groups by one-way analysis of variance (ANOVA).

Results
Establishment of the high-throughput system and first screening for β-TrCP ligands C-IκB-PP15 is a phosphopeptide whose sequence is derived from IκB that is known to bind to β-TrCP. C-IκB-s15 is its non-phosphorylated version, which cannot bind to β-TrCP and was used as a negative control. They were both bound to the maleimide-activated plate using N-terminal cysteine. Express SF+ cell lysates expressing mAG-β-TrCP1 were added into the 96-well plate. After enough binding and washing, the bound mAG-β-TrCP1 was quantitated using spectrofluorometry. The postwash value indicates the binding of mAG-β-TrCP1 and C-IκB-PP15. When the small molecules compete with the phosphopeptides for mAG-β-TrCP1 binding, there was less plate fluorescence (Fig. 1A).
The free target phosphopeptide competitively inhibited the binding of the mAG-β-TrCP1 to the plate-fixed phosphopeptide. The efficacy of the system was confirmed by mixing serially diluted phosphopeptide into the cell lysate (Fig. 1B). To identify potent ligands of β-TrCP, 5,000 RIKEN Natural Products Depository (NPDepo) compounds were examined using this high-throughput screening system in a final concentration of 0.2 mg/mL and 109 small molecules were identified as initial positive compounds inhibiting binding by >~20% (Fig. 1C). Then, 109 compounds were re-screened at a lower concentration (0.2 mM), and 13 hits were obtained. Furthermore, 372 derivatives of these 13 initial hits and 27 derivatives of known inhibitor GS143 were examined. Finally, 23 positive NPDepo compounds (13 hits + 10 positive derivatives) and 13 positive GS143 derivatives were identified from the highthroughput system.
The secondary screening-in vitro ubiquitination assay As the secondary screening, the activity of hit compounds was examined using the SCF β-TrCP1 ubiquitination activity in vitro at a final concentration of 100 µM. The kinase-negative mutant GST-Wee1 KR was used as the substrate of SCF β-TrCP1 .
Effect of the hit compounds on Wnt/β-catenin signaling pathway In the classic Wnt signaling pathway, when β-TrCP induced ubiquitination is inhibited, β-catenin accumulates and further activates nucleolar TCF transcription [31,32]. We examined the effects of hit compounds on β-catenin degradation in HeLa cells. HeLa cells were cultured for 3 h with or without the compound. The levels of β-catenin and p-β-catenin were examined in the cell extracts. Proteasome inhibitor MG132 caused the accumulation of β-catenin and phosphorylated form, while the GS143 did not affect βcatenin and p-β-catenin, which was similar to the published results [8]. Among the seven hit compounds identified from the screening above, only GS143 derivatives NPL72038-01 and NPL62039-01 promoted the accumulation of phosphorylated β-catenin. However, they did not significantly affect the β-catenin level (Fig. 3A).
Wnt/β-catenin signaling pathway activity was also assessed using TOP/FOP-Flash luciferase reporter assay. The average ratio of TOP to renilla luciferase divided by FOP was used as TCF activity. Wnt3a is the molecule that stimulates classic Wnt signaling [11]. After adding Wnt3a, the level of TCF transcription increased, which confirmed the efficacy of this system (Fig. 3B). Similarly, in the immunoblotting result obtained, GS143 did not affect TCF transcription and was inhibited minimally. NPL72038-01 and NPL62039-01 also did not cause a high increase in TCF transcription (Fig. 3C).
Effect of the hit compounds on the NF-kB signaling pathway NF-κB is considered a typical pro-inflammatory signaling pathway, mediated by the effect of pro-inflammatory cytokines like TNF-α. TNF-α induces the β-TrCPdependent ubiquitination and degradation of IκBα and relocates NF-κB into the nucleus [33,34]. The impacts of hit compounds on IκBα degradation were further examined in HeLa cells. Cells were cultured for 30 min with or without the indicated compounds. Then, TNF-α was added and incubated for an extra 5 min. The level of IκBα and p-IκBα was examined in cell extracts. MG132 and GS143 caused the inhibition of the degradation of IκBα and its phosphorylated form, just as published [34]. Among the seven hit compounds from secondary screening, NPDepo compound NPD5943 and GS143 derivatives NPL72038-01, NPL62020-01, and NPL42040-01 increased the level of IκBα and p-IκBα (Fig. 4A).
NF-κB transcription activity was also determined by firefly luciferase expression normalized by renilla luciferase level. TNF-α activated the transcription of NF-κB. Among (The data are presented as the average ± S.D. The statistical differences among the groups were evaluated using one-way ANOVA. p-value: **** < 0.0001) (C) Results of initial screening of~5,000 compounds. The effect of each compound (at 0.2 mg/mL) on mAG-β-TrCP1 binding was standardized and is represented as a percentage value of control (100% indicating no inhibition). The compounds that inhibited the binding by >20% were the initial hits. the seven potent compounds from the secondary screening, the treatment of NPD5943, NPL62020-01, and NPL42040-01 reduced NF-κB activation dose-dependently. NPL82037-01 and NPD945 also caused accumulation of TNF-α induced IκBα and p-IκBα level. However, NPL82037-01 caused a high cell death at high concentrations, and NPD945 did not affect NF-κB transcription activity. These two compounds were not regarded as effective in NF-kB signaling pathway. GS143 at 20 μM inhibited the transcription to~40% of that of the control group (DMSO, TNF-α). NPD5943 and NPL62020-01 can inhibit it to approximately 20% of that of the control at 20 μM, which shows better inhibition than GS143 at high concentration (Fig. 4B).
The compounds that exhibit inhibitory effect on either the β-catenin or NF-κB pathways illustrate their binding ability to β-TrCP E3 ligase, and can be considered as hit ligands. Therefore, although these compounds (NPD5943, NPL42040-01, and NPL62020-01) do not have noticeable effects on the β-catenin pathway, we still regarded them as the positive ligands because of their significant effects on the NF-kB pathway. However, NPL72038-01 and NPL62039-01 did not exhibit significant effect in either β-catenin or NF-kB signaling pathway; hence, we did not regard them as hit compounds.
In silico analysis of the hit compounds The structures of the three hit compounds were further analyzed using in silico analysis to examine the important moieties and explain their activity. Molecular docking and binding site analysis of β-TrCP (PDB:1P22) were carried out with hit compounds using the docking program autodock vina and PyMOL software. The results were achieved by extensively using autodock tools for setting up docking runs. The meta information, including the docking score, is displayed for each pose in an automatically recorded text Reportedly, β-TrCP recognizes a specific motif with phosphorylated serines [7]. Their interface indicated the hydrogen bond (H bond) binding to the phosphorylated site. The amino acids bound to pS33 (Y271, R285, S309, and S325) are marked in a yellow circle, and the amino acids bound to pS37 (S448, R431, and G432) are marked in a white circle (Fig. 5A). We focused on these amino acids for the H bond formation with the compounds.
The result shows GS143 created three H bonds with β-TrCP. One binding amino acid of the H bond created by carboxyl groups is similar to β-catenin pS33 binding amino acid (Y271), indicating the importance of the carboxyl groups. By comparing the structures of the three hit compounds, we noticed that the carboxyl groups of the two GS143 derivatives and the hydroxyl group of NPD5943 created H bonds when bound to β-TrCP. The two active GS143 derivatives created more H bonds than GS143, and they have the same binding sites with both pS33 and pS37 (NPL42040-01: Y271, S325, S448, R431; NPL62020-01: Y271, S325, S448). Among them, NPL42040-01 is the most effective in immunoblotting results; it created a total of six H bonds in the binding, and four of their binding residues belong to both pS33 and pS37 (Fig. 5B).
To further explore the importance of carboxyl groups, we analyzed another negative ligand (NPL72029-01) that does not have the carboxyl groups. The result shows that NPL72029-01 did not create any H bonds to β-TrCP, which indicates the importance of carboxyl to β-TrCP binding.

Discussion
A significant level of SCF β-TrCP is available in the cells, showing enormous potential to be a target of PROTAC [31]. β-TrCP can potentially be a competitive target of the PROTAC technique. A sensitive and rapid screening system may provide more possibilities in the synthesis.
In this study, we established a high-throughput screening system by constructing recombinant baculoviruses to express fluorescent fusion protein mAG-β-TrCP1 and identified the ligands of β-TrCP with it. The high-throughput screening revealed 36 compounds that show competitive binding activity to β-TrCP. In vitro ubiquitination assay re-examined these compounds; seven were obtained as hit compounds. The effect of the hit compounds on substrate β-catenin and IκBα degradation was further examined to reflect the competitive binding activity to β-TrCP.
Results revealed that a majority of the hit compounds caused marked intracellular accumulation of IκBα and its phosphorylated form, similar to the effect of the proteasome inhibitor MG132. Three of the hit compounds showed an inhibitory effect on TNF-α induced NF-κB transcription in a dose-dependent manner, which proved their effectiveness in the NF-κB signaling pathway. As GS143 is a specific inhibitor of β-TrCP [8], NPL62020-01 and NPL42040-01 were also predicted to be specific to β-TrCP. As for NPD5943, we examined its binding to another F-Box protein of SCF-type E3 ligase, SKP2. As expected, NPD5943 did not bind to SKP2 at all (data not shown). In addition, because β-TrCP binds to phosphorylated peptides, it is possible that its ligand can bind to other proteins that bind to phosphorylated proteins. The polo-box domain of Plk1, 14-3-3 proteins, and Pin1 are examples of such protein.
When we examined the binding of NPD5943 to such proteins, we did not observe any binding (data not shown). These results suggest that the binding of NPD5943 to β-TrCP is highly specific. We also examined the non-specific cytotoxicity of NPD5943, NPL62020-01, and NPL42040-01. We found that these compounds showed cytotoxicity only at high concentration (data not shown). As inhibition of β-TrCP by siRNA did not show any cytotoxicic effects on HeLa cells, these results also support the fact that these hit compounds do not have any non-specific inhibition to other proteins.
A majority of the hit compounds that affect the degradation of IkBα did not result in the accumulation of βcatenin, the E3 of which is identical to the SCF β-TrCP1 of IκBα. Moreover, the two hit compounds that inhibited the p-β-catenin degradation did not increase β-catenin itself, indicating that only a small population of β-catenin was increased by these compounds. We speculate that this increase may not be sufficient to promote TCF transcription. Thus, we could not find compounds that inhibited the β-catenin degradation.
Corroborating with findings of a previous study, GS143 also showed similar behavior that explicitly targets IkBα degradation, excluding β-catenin [8]. GS143 was identified by examining the IκBα ubiquitination using TR-FRET. This effect of GS143 was more likely to be a physical interaction with p-IκBα and SCF β-TrCP1 , the disruption of which inhibits IκBα ubiquitination [8]. However, in this study, these compounds were screened using binding and in vitro ubiquitination assays, which confirmed their competitive binding activity to β-TrCP. We hypothesized that such an effect may be because their chemical moieties result in different binding characters. GS143 has been reported as a novel NF-κB signaling inhibitor with great potential in the treatment of airway inflammation in asthmatic patients [34]. Here, we found more potent compounds that function better than GS143 in the inhibition of TNF-α induced IkBα degradation and the activation of NF-κB. The hit compounds obtained here showed potent effect in NF-κB signaling, and did not increase the level of β-catenin, which can be candidate compounds with anti-inflammatory and anti-tumor properties. This possibility can be explored in future studies.
In silico analysis summarized the important moieties and explained why the three hit compounds are active. According to the results, when GS143 binds to β-TrCP, the carboxyl moiety of GS143 has two hydrogen bonds; one had the same occupation as the amino acid of β-TrCP that was created with β-catenin pS33. The carboxyl moiety of the two GS143 derivatives also created hydrogen bonds when bound to β-TrCP, while the negative compound without carboxyl FIGURE 5. In silico analysis of the hit compounds. (A) The interface of phosphorylated β-catenin (pS33, pS37) and β-TrCP: β-TrCP is shown in red, with its side chains in pink, and the phosphodegron peptides of β-catenin are in yellow (phosphate groups on serines are shown in green and red). White dotted lines represent H bonds. Amino acid residues linked to pS33 of β-catenin and those to pS37 are marked in yellow and white circles, respectively. These phosphoamino acids of β-catenin, pS33 and pS37 are marked in yellow and white boxes, respectively. The original figure from [7] was modified. (B) The interface of hit compounds and β-TrCP: β-TrCP is shown in light blue. When amino acids shown in (A) create an H bond with the compound, they are shown in the same color as in (A). (C) The interface of negative compound (NPL72029-01) and β-TrCP: β-TrCP is shown in light blue. The structure of NPL72029-01 is shown on the right. groups did not. These results may explain its importance in these GS143 derivatives. Hydroxyl groups of NPD5943 also play an important role in H bond creation. These residues should be protected in the synthesis of PROTAC. All the two active GS143 derivatives have binding residues that belong to pS33 and pS37. We presumed GS143 derivatives with more H bond occupations of pS33 and pS37 binding amino acids may show high activity in β-TrCP binding.
In summary, we developed a flexible, sensitive and, specific system that can be used to screen β-TrCP ligands. The potent ligands for β-TrCP were finally obtained as expected, which can be used as tools for PROTAC construction in the future. PROTAC technology has significant advantages over conventional drugs, and has shown great potential as a therapeutic and biological tool. This study provides a new prospect of the target E3 ligase selection and promising binding ligands of PROTACs construction. The potent ligands can be used as elements of the PROTAC complex that recruits β-TrCP to the protein of interest, which may prove great potentiality in clinic therapy in the future.